RESUMEN
Signaling via the transient receptor potential (TRP) ion channel C6 plays a pivotal role in hereditary and sporadic glomerular kidney disease. Several studies have identified gain-of-function mutations of TRPC6 and report induced expression and enhanced channel activity of TRPC6 in association with glomerular diseases. Interfering with TRPC6 activity may open novel therapeutic pathways. TRPC6 channel activity is controlled by protein expression and stability as well as intracellular trafficking. Identification of regulatory phosphorylation sites in TRPC6 and corresponding protein kinases is essential to understand the regulation of TRPC6 activity and may result in future therapeutic strategies. In this study, an unbiased phosphoproteomic screen of human TRPC6 identified several novel serine phosphorylation sites. The phosphorylation site at serine 14 of TRPC6 is embedded in a basophilic kinase motif that is highly conserved across species. We confirmed serine 14 as a target of MAPKs and proline-directed kinases like cyclin-dependent kinase 5 (Cdk5) in cell-based as well as in vitro kinase assays and quantitative phosphoproteomic analysis of TRPC6. Phosphorylation of TRPC6 at serine 14 enhances channel conductance by boosting membrane expression of TRPC6, whereas protein stability and multimerization of TRPC6 are not altered, making serine 14 phosphorylation a potential drug target to interfere with TRPC6 channel activity.-Hagmann, H., Mangold, N., Rinschen, M. M., Koenig, T., Kunzelmann, K., Schermer, B., Benzing, T., Brinkkoetter, P. T. Proline-dependent and basophilic kinases phosphorylate human TRPC6 at serine 14 to control channel activity through increased membrane expression.
Asunto(s)
Proteínas Quinasas Dirigidas por Prolina/metabolismo , Canal Catiónico TRPC6/metabolismo , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Secuencia Conservada , Quinasa 5 Dependiente de la Ciclina/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Femenino , Células HEK293 , Células HeLa , Humanos , Técnicas In Vitro , Oocitos/metabolismo , Fosforilación , Estabilidad Proteica , Proteómica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Canal Catiónico TRPC6/química , Canal Catiónico TRPC6/genética , Xenopus laevisRESUMEN
BACKGROUND: Acute kidney injury (AKI) is a common complication after cardiopulmonary resuscitation (CPR) and predicts in-hospital mortality. To which extent post-resuscitation disease or the initial event of cardiac arrest and the duration of insufficient cardiac output triggers AKI is challenging to discriminate. Knowledge on molecular mediators of AKI is scarce. Early identification of patients at high risk of AKI is hampered by the low sensitivity of the established tests in clinical routine practice. The present study aimed to determine the diagnostic utility of the novel urine biomarkers tissue inhibitor of metalloproteinases-2 (TIMP-2) and insulin-like growth factor-binding protein 7 (IGFBP7) for the early recognition of AKI in patients with non-traumatic shock. METHODS: The performance of [TIMP-2]·[IGFBP7] was prospectively analysed in 48 patients with shock following out-of-hospital cardiac arrest (OHCA). All patients were treated with target temperature management (TTM) for 24 h. Urinary [TIMP-2]·[IGFBP7] samples were collected at 3 and 24 h after determination of OHCA. RESULTS: Patients (n = 31 (65%)) developed AKI after an average of 26 ± 12 h. Patients who developed AKI had significantly higher [TIMP-2]·[IGFBP7] compared to individuals that did not develop AKI (1.52 ± 0.13 vs. 0.13 ± 0.14; p < 0.05) as early as 3 h after determination of OHCA,. For urine [TIMP-2]*[IGFBP7], the area under the curve (AUC) for the development of AKI was 0.97 (CI 0.90-1.00) at 3 h after OHCA. The optimal [TIMP-2]·[IGFBP7] cut-off value for the prediction of AKI was 0.24. The sensitivity was 96.8% and specificity was 94.1%. CONCLUSIONS: Urinary [TIMP-2]â¢[IGFBP7] reliably predicts AKI in high-risk patients only 3 h after determination of OHCA with a cut-off at 0.24. This novel test may help to identify patients at high risk of AKI to enrol into clinical studies to further elucidate the pathophysiology of AKI and devise targeted interventions in the future.
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Lesión Renal Aguda/sangre , Paro Cardíaco Extrahospitalario/complicaciones , Sobrevivientes/estadística & datos numéricos , Lesión Renal Aguda/mortalidad , Lesión Renal Aguda/fisiopatología , Anciano , Área Bajo la Curva , Biomarcadores/análisis , Biomarcadores/sangre , Femenino , Humanos , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/análisis , Proteínas de Unión a Factor de Crecimiento Similar a la Insulina/sangre , Masculino , Persona de Mediana Edad , Paro Cardíaco Extrahospitalario/sangre , Paro Cardíaco Extrahospitalario/fisiopatología , Valor Predictivo de las Pruebas , Curva ROC , Inhibidor Tisular de Metaloproteinasa-2/análisis , Inhibidor Tisular de Metaloproteinasa-2/sangreRESUMEN
Podocyte injury is a key event in glomerular disease leading to proteinuria and opening the path toward glomerular scarring. As a consequence, glomerular research strives to discover molecular mechanisms and signaling pathways affecting podocyte health. The hNphs2.Cre mouse model has been a valuable tool to manipulate podocyte-specific genes and to label podocytes for lineage tracing and purification. Here we designed a novel podocyte-specific tricistronic Cre mouse model combining codon improved Cre expression and fluorescent cell labeling with mTomato under the control of the endogenous Nphs2 promoter using viral T2A-peptides. Independent expression of endogenous podocin, codon improved Cre, and mTomato was confirmed by immunofluorescence, fluorescent activated cell sorting and protein analyses. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice developed normally and did not show any signs of glomerular disease or off-target effects under basal conditions and in states of disease. Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type-mediated gene recombination was superior to conventional hNphs2.Cre mice-mediated gene recombination. Last, we compared Cre efficiency in a disease model by mating Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type and hNphs2.Cre mice to Phb2fl/fl mice. The podocyte-specific Phb2 knockout by Nphs2pod.T2A.ciCre.T2A.mTomato/wild-type mice resulted in an aggravated glomerular injury as compared to a podocyte-specific Phb2 gene deletion triggered by hNphs2.Cre. Thus, we generated the first tricistronic podocyte mouse model combining enhanced Cre recombinase efficiency and fluorescent labeling in podocytes without the need for additional matings with conventional reporter mouse lines.
Asunto(s)
Técnicas de Sustitución del Gen , Genes Reporteros , Integrasas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Luminiscentes/genética , Proteínas de la Membrana/genética , Péptidos/genética , Podocitos/metabolismo , Proteínas Virales/genética , Animales , Separación Celular/métodos , Codón , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Regulación de la Expresión Génica , Péptidos y Proteínas de Señalización Intracelular/biosíntesis , Proteínas Luminiscentes/biosíntesis , Proteínas de la Membrana/biosíntesis , Ratones Endogámicos C57BL , Ratones Transgénicos , Prohibitinas , Regiones Promotoras Genéticas , Factores de Tiempo , Proteína Fluorescente RojaAsunto(s)
Complicaciones Hematológicas del Embarazo/tratamiento farmacológico , Púrpura Trombocitopénica Trombótica/tratamiento farmacológico , Anticuerpos de Dominio Único/uso terapéutico , Factor de von Willebrand/antagonistas & inhibidores , Proteína ADAMTS13/inmunología , Manejo de la Enfermedad , Femenino , Humanos , Embarazo , Complicaciones Hematológicas del Embarazo/diagnóstico , Complicaciones Hematológicas del Embarazo/etiología , Resultado del Embarazo , Púrpura Trombocitopénica Trombótica/diagnóstico , Púrpura Trombocitopénica Trombótica/etiología , Anticuerpos de Dominio Único/administración & dosificación , Anticuerpos de Dominio Único/efectos adversos , Resultado del TratamientoRESUMEN
Mitochondrial fusion is essential for maintenance of mitochondrial function and requires the prohibitin ring complex subunit prohibitin-2 (PHB2) at the mitochondrial inner membrane. Loss of the stomatin/PHB/flotillin/HflK/C (SPFH) domain containing protein PHB2 causes mitochondrial dysfunction and defective mitochondria-mediated signaling, which is implicated in a variety of human diseases, including progressive renal disease. Here, we provide evidence of additional, extra-mitochondrial functions of this membrane-anchored protein. Immunofluorescence and immunogold labeling detected PHB2 at mitochondrial membranes and at the slit diaphragm, a specialized cell junction at the filtration slit of glomerular podocytes. PHB2 coprecipitated with podocin, another SPFH domain-containing protein, essential for the assembly of the slit diaphragm protein-lipid supercomplex. Consistent with an evolutionarily conserved extra-mitochondrial function, the ortholog of PHB2 in Caenorhabditis elegans was also not restricted to mitochondria but colocalized with the mechanosensory complex that requires the podocin ortholog MEC2 for assembly. Knockdown of phb-2 partially phenocopied loss of mec-2 in touch neurons of the nematode, resulting in impaired gentle touch sensitivity. Collectively, these data indicate that, besides its established role in mitochondria, PHB2 may have an additional function in conserved protein-lipid complexes at the plasma membrane.
Asunto(s)
Mitocondrias/fisiología , Podocitos/fisiología , Proteínas Represoras/deficiencia , Animales , Proteínas de Caenorhabditis elegans , Células Cultivadas , Células HEK293 , Humanos , Uniones Intercelulares/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Riñón/fisiología , Mecanorreceptores/fisiología , Mecanotransducción Celular/fisiología , Proteínas de la Membrana/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica , Mitocondrias/ultraestructura , Enfermedades Mitocondriales/etiología , Enfermedades Mitocondriales/fisiopatología , Membranas Mitocondriales/fisiología , Membranas Mitocondriales/ultraestructura , Podocitos/ultraestructura , Prohibitinas , Proteinuria/etiología , Proteinuria/fisiopatología , Tacto/fisiologíaRESUMEN
Preeclampsia is a devastating complication of pregnancy. Soluble Fms-like tyrosine kinase-1 (sFlt-1) is an antiangiogenic protein believed to mediate the signs and symptoms of preeclampsia. We conducted an open pilot study to evaluate the safety and potential efficacy of therapeutic apheresis with a plasma-specific dextran sulfate column to remove circulating sFlt-1 in 11 pregnant women (20-38 years of age) with very preterm preeclampsia (23-32 weeks of gestation, systolic BP ≥140 mmHg or diastolic BP ≥90 mmHg, new onset protein/creatinine ratio >0.30 g/g, and sFlt-1/placental growth factor ratio >85). We evaluated the extent of sFlt-1 removal, proteinuria reduction, pregnancy continuation, and neonatal and fetal safety of apheresis after one (n=6), two (n=4), or three (n=1) apheresis treatments. Mean sFlt-1 levels were reduced by 18% (range 7%-28%) with concomitant reductions of 44% in protein/creatinine ratios. Pregnancy continued for 8 days (range 2-11) and 15 days (range 11-21) in women treated once and multiple times, respectively, compared with 3 days (range 0-14) in untreated contemporaneous preeclampsia controls (n=22). Transient maternal BP reduction during apheresis was managed by withholding pre-apheresis antihypertensive therapy, saline prehydration, and reducing blood flow through the apheresis column. Compared with infants born prematurely to untreated women with and without preeclampsia (n=22 per group), no adverse effects of apheresis were observed. In conclusion, therapeutic apheresis reduced circulating sFlt-1 and proteinuria in women with very preterm preeclampsia and appeared to prolong pregnancy without major adverse maternal or fetal consequences. A controlled trial is warranted to confirm these findings.
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Peso al Nacer , Eliminación de Componentes Sanguíneos/métodos , Sulfato de Dextran/uso terapéutico , Preeclampsia/terapia , Receptor 1 de Factores de Crecimiento Endotelial Vascular/sangre , Adulto , Eliminación de Componentes Sanguíneos/efectos adversos , Presión Sanguínea , Sulfato de Dextran/química , Femenino , Edad Gestacional , Frecuencia Cardíaca Fetal , Humanos , Recién Nacido , Terapia por Inhalación de Oxígeno , Proyectos Piloto , Preeclampsia/sangre , Embarazo , Mantenimiento del Embarazo , Nacimiento Prematuro/prevención & control , Proteinuria/terapia , Receptor 1 de Factores de Crecimiento Endotelial Vascular/química , Adulto JovenRESUMEN
Following genotoxic stress, cells activate a complex signalling network to arrest the cell cycle and initiate DNA repair or apoptosis. The tumour suppressor p53 lies at the heart of this DNA damage response. However, it remains incompletely understood, which signalling molecules dictate the choice between these different cellular outcomes. Here, we identify the transcriptional regulator apoptosis-antagonizing transcription factor (AATF)/Che-1 as a critical regulator of the cellular outcome of the p53 response. Upon genotoxic stress, AATF is phosphorylated by the checkpoint kinase MK2. Phosphorylation results in the release of AATF from cytoplasmic MRLC3 and subsequent nuclear translocation where AATF binds to the PUMA, BAX and BAK promoter regions to repress p53-driven expression of these pro-apoptotic genes. In xenograft experiments, mice exhibit a dramatically enhanced response of AATF-depleted tumours following genotoxic chemotherapy with adriamycin. The exogenous expression of a phospho-mimicking AATF point mutant results in marked adriamycin resistance in vivo. Nuclear AATF enrichment appears to be selected for in p53-proficient endometrial cancers. Furthermore, focal copy number gains at the AATF locus in neuroblastoma, which is known to be almost exclusively p53-proficient, correlate with an adverse prognosis and reduced overall survival. These data identify the p38/MK2/AATF signalling module as a critical repressor of p53-driven apoptosis and commend this pathway as a target for DNA damage-sensitizing therapeutic regimens.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/fisiología , Apoptosis/fisiología , Daño del ADN/fisiología , Proteínas Represoras/fisiología , Proteína p53 Supresora de Tumor/fisiología , Transporte Activo de Núcleo Celular , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/genética , Puntos de Control del Ciclo Celular , Daño del ADN/genética , Doxorrubicina/farmacología , Resistencia a Antineoplásicos/genética , Neoplasias Endometriales/genética , Femenino , Amplificación de Genes , Dosificación de Gen , Células HEK293 , Humanos , Ratones , Datos de Secuencia Molecular , Complejos Multiproteicos , Cadenas Ligeras de Miosina/metabolismo , Neuroblastoma/genética , Neuroblastoma/mortalidad , Presión Osmótica , Fosforilación , Pronóstico , Procesamiento Proteico-Postraduccional , Proteínas Represoras/genéticaAsunto(s)
Adenosina Monofosfato/análogos & derivados , Alanina/análogos & derivados , Antivirales/sangre , COVID-19/sangre , Fallo Renal Crónico/sangre , Diálisis Renal/tendencias , Adenosina Monofosfato/administración & dosificación , Adenosina Monofosfato/sangre , Adenosina Monofosfato/farmacocinética , Anciano , Alanina/administración & dosificación , Alanina/sangre , Alanina/farmacocinética , Antivirales/administración & dosificación , Antivirales/farmacocinética , COVID-19/complicaciones , Humanos , Infusiones Intravenosas/métodos , Fallo Renal Crónico/complicaciones , Fallo Renal Crónico/tratamiento farmacológico , Masculino , Tratamiento Farmacológico de COVID-19RESUMEN
BACKGROUND: Steroid-resistant nephrotic syndrome (SRNS) is a severe cause of progressive renal disease. Genetic forms of SRNS can present with autosomal recessive or autosomal dominant inheritance. Recent studies have identified mutations in multiple podocyte genes responsible for SRNS. Improved sequencing methods (next-generation sequencing, NGS) now promise rapid mutational testing of SRNS genes. METHODS: In the present study, a simultaneous screening of ten SRNS genes in 37 SRNS patients was performed by NGS. RESULTS: In 38 % of the patients, causative mutations in one SRNS gene were found. In 22 % of the patients, in addition to these mutations, a secondary variant in a different gene was identified. CONCLUSIONS: This high incidence of accumulating sequence variants was unexpected but, although they might have modifier effects, the pathogenic potential of these additional sequence variants seems unclear so far. The example of molecular diagnostics by NGS in SRNS patients shows that these new sequencing technologies might provide further insight into molecular pathogenicity in genetic disorders but will also generate results, which will be difficult to interpret and complicate genetic counseling. Although NGS promises more frequent identification of disease-causing mutations, the identification of causative mutations, the interpretation of incidental findings and possible pitfalls might pose problems, which hopefully will decrease by further experience and elucidation of molecular interactions.
Asunto(s)
Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Hallazgos Incidentales , Técnicas de Diagnóstico Molecular , Mutación , Síndrome Nefrótico/congénito , Adolescente , Niño , Preescolar , Femenino , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Humanos , Lactante , Masculino , Persona de Mediana Edad , Síndrome Nefrótico/diagnóstico , Síndrome Nefrótico/genética , Fenotipo , Valor Predictivo de las Pruebas , Pronóstico , Adulto JovenRESUMEN
The atypical cyclin-dependent kinase 5 (Cdk5) serves an array of different functions in cell biology. Among these are axonal guidance, regulation of intercellular contacts, cell differentiation, and prosurvival signaling. The variance of these functions suggests that Cdk5 activation comes to pass in different cellular compartments. The kinase activity, half-life, and substrate specificity of Cdk5 largely depend on specific activators, such as p25, p35, p39, and cyclin I. We hypothesized that the subcellular distribution of Cdk5 activators also determines the localization of the Cdk5 protein and sets the stage for targeted kinase activity within distinct cellular compartments to suit the varying roles of Cdk5. Cdk5 localization was analyzed in murine kidney and brain slices of wild-type and cyclin I- and/or p35-null mice by immunohistochemistry and in cultured mouse podocytes using immunofluorescence labeling, as well as cell fractionation experiments. The predominance of cyclin I mediates the nuclear localization of Cdk5, whereas the predominance of p35 results in a membranous localization of Cdk5. These findings were further substantiated by overexpression of cyclin I and p35 with altered targeting characteristics in human embryonic kidney 293T cells. These studies reveal that the subcellular localization of Cdk5 is determined by its specific activators. This results in the directed Cdk5 kinase activity in specific cellular compartments dependent on the activator present and allows Cdk5 to serve multiple independent roles.
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Ciclina I/metabolismo , Quinasa 5 Dependiente de la Ciclina/metabolismo , Fosfotransferasas/metabolismo , Podocitos/enzimología , Animales , Membrana Celular/enzimología , Núcleo Celular/enzimología , Ciclina I/deficiencia , Ciclina I/genética , Retículo Endoplásmico/enzimología , Activación Enzimática , Células HEK293 , Humanos , Ratones Noqueados , Fosfotransferasas/deficiencia , Fosfotransferasas/genética , Transporte de Proteínas , Células de Purkinje/enzimología , TransfecciónRESUMEN
TNF has been implicated in glomerular diseases, but its actions on podocytes are not well understood. Endogenous TNF expression is markedly increased in mouse podocytes exposed to sera from patients with recurrent focal segmental glomerulosclerosis, and TNF is able to increase its own expression in these cells. Exposure of podocytes to TNF increased phosphorylation of NF-κB p65-RelA followed by increased tyrosine phosphorylation of STAT3. STAT3 activation was blocked by the NF-κB inhibitor JSH-23 and by the STAT3 inhibitor stattic, whereas TNF-evoked NF-κB activation was not affected by stattic. TNF treatment increased nuclear accumulation of nuclear factor of activated T cells (NFAT)c1 in podocytes, a process that occurred downstream of STAT3 activation. TNF also increased expression of cyclin D1 but had no effect on cyclin-dependent kinase 4, p27(kip), or podocin. Despite its effects on cyclin D1, TNF treatment for up to 72 h did not cause podocytes to reenter the cell cycle. TNF increased total expression of transient receptor potential (TRP)C6 channels through a pathway dependent on NFATc1 and increased the steady-state expression of TRPC6 subunits on the podocyte cell surface. TNF effects on TRPC6 trafficking required ROS. Consistent with this, La(3+)-sensitive cationic currents activated by a diacylglycerol analog were increased in TNF-treated cells. The effects of TNF on NFATc1 and TRPC6 expression were blocked by cyclosporine A but were not blocked by the pan-TRP inhibitor SKF-96365. TNF therefore influences multiple pathways previously implicated in podocyte pathophysiology and is likely to sensitize these cells to other insults.
Asunto(s)
Podocitos/metabolismo , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Animales , Línea Celular , Glomeruloesclerosis Focal y Segmentaria/metabolismo , Humanos , Masculino , Ratones , Factores de Transcripción NFATC/metabolismo , Factor de Transcripción STAT3/metabolismo , Canales Catiónicos TRPC/metabolismo , Canal Catiónico TRPC6RESUMEN
Lipid peroxidation through electrophilic molecules of extracellular origin is involved in the pathogenesis of many inflammatory conditions. To counteract free radical actions at the plasma membrane, cells host a variety of antioxidative enzymes. Here we analyzed localization, membrane topology, and trafficking of PON2 a member of the paraoxonase family of 3 enzymatically active proteins (PON1-3) found to have antiatherogenic properties. Immunohistochemistry localized PON2 to the villous tip of human intestinal epithelial cells. Employing membrane preparations, surface biotinylation experiments, and mutational analyses in HEK 293T and HeLa cells, we demonstrate that PON2 is a type II transmembrane protein. A hydrophobic stretch in the N terminus was identified as single transmembrane domain of PON2. The enzymatically active domain faced the extracellular compartment, where it suppressed lipid peroxidation (P<0.05) and regulated the glucosylceramide content, as demonstrated by mass spectrometry (P<0.05). PON2 translocation to the plasma membrane was dependent on intracellular calcium responses and could be induced to >10-fold as compared to baseline (P=0.0001) by oxidative stress. Taken together, these data identify the paraoxonase protein PON2 as a type II transmembrane protein, which is dynamically translocated to the plasma membrane in response to oxidative stress to counteract lipid peroxidation.
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Arildialquilfosfatasa/metabolismo , Membrana Celular/metabolismo , Peroxidación de Lípido , Proteínas de la Membrana/metabolismo , Secuencia de Aminoácidos , Animales , Arildialquilfosfatasa/genética , Calcio/metabolismo , Células Epiteliales/enzimología , Glucosilceramidas/metabolismo , Células HEK293 , Células HeLa , Humanos , Immunoblotting , Inmunohistoquímica , Intestinos/citología , Intestinos/enzimología , Proteínas de la Membrana/genética , Ratones , Microscopía Confocal , Datos de Secuencia Molecular , Estrés Oxidativo , Transporte de Proteínas , Interferencia de ARNRESUMEN
Diseases of the kidney filtration barrier are a leading cause of ESRD. Most disorders affect the podocytes, polarized cells with a limited capacity for self-renewal that require tightly controlled signaling to maintain their integrity, viability, and function. Here, we provide an atlas of in vivo phosphorylated, glomerulus-expressed proteins, including podocyte-specific gene products, identified in an unbiased tandem mass spectrometry-based approach. We discovered 2449 phosphorylated proteins corresponding to 4079 identified high-confidence phosphorylated residues and performed a systematic bioinformatics analysis of this dataset. We discovered 146 phosphorylation sites on proteins abundantly expressed in podocytes. The prohibitin homology domain of the slit diaphragm protein podocin contained one such site, threonine 234 (T234), located within a phosphorylation motif that is mutated in human genetic forms of proteinuria. The T234 site resides at the interface of podocin dimers. Free energy calculation through molecular dynamic simulations revealed a role for T234 in regulating podocin dimerization. We show that phosphorylation critically regulates formation of high molecular weight complexes and that this may represent a general principle for the assembly of proteins containing prohibitin homology domains.
Asunto(s)
Barrera de Filtración Glomerular/fisiología , Fosfoproteínas/análisis , Fosfoproteínas/fisiología , Proteómica , Animales , Femenino , Péptidos y Proteínas de Señalización Intracelular/fisiología , Proteínas de la Membrana/fisiología , Ratones , Fosforilación , Podocitos/fisiologíaRESUMEN
Podocytes, the visceral epithelial cells of the kidney glomerulus, elaborate primary and interdigitating secondary extensions to enwrap the glomerular capillaries. A hallmark of podocyte injury is the loss of unique ultrastructure and simplification of the cell shape, called foot process effacement, which is a classic feature of proteinuric kidney disease. Although several key pathways have been identified that control cytoskeletal regulation, actin dynamics, and polarity signaling, studies into the dynamic regulation of the podocyte structure have been hampered by the fact that ultrastructural analyses require electron microscopic imaging of fixed tissue. We developed a new technique that allows for visualization of podocyte foot processes using confocal laser scanning microscopy. The combination of inducible and mosaic expression of membrane-tagged fluorescent proteins in a small subset of podocytes enabled us to acquire light microscopic images of podocyte foot processes in unprecedented detail, even in living podocytes of freshly isolated glomeruli. Moreover, this technique visualized oscillatory glomerular contractions and confirmed the morphometric evaluations obtained in static electron microscopic images of podocyte processes. These data suggest that the new technique will provide an extremely powerful tool for studying the dynamics of podocyte ultrastructure.
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Podocitos/citología , Podocitos/ultraestructura , Animales , Cruzamientos Genéticos , Modelos Animales de Enfermedad , Femenino , Proteínas Fluorescentes Verdes/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mosaicismo , Podocitos/fisiologíaRESUMEN
Idiopathic inflammatory myopathy (IIM) summarizes rare, systemic autoimmune conditions primarily characterized by inflammatory damage to the skeletal muscle. Although primary damage occurs to the muscle, these IIM-related conditions involve other organs, including the skin, lungs, upper gastrointestinal tract, joints, and heart. While many patients have an adequate response to immunosuppressive treatment, some patients develop rapidly progressive and treatment-resistant life-threatening courses. Treatment-resistant IIM is challenging for the treating physician and requires interdisciplinary and individualized treatment approaches. Extracorporeal therapy is one option for rescue therapy, with immunoadsorption (IA) having proven more effective than plasma exchange regarding the removal of circulating antibodies. Despite its efficacy and desirable safety profile, the clinical value of IA use in IIM is understudied with no controlled trials reported. Here, we present a review of the current knowledge regarding the management of treatment-resistant IIM and the cases of three patients with treatment-resistant IIM (two with dermatomyositis and one with immune-mediated necrotizing myopathy) who have successfully been treated with IA. All patients responded well to the therapy and experienced no IA-related complications. Taken together, we found IA to be a safe and effective treatment option in treatment-resistant IIM.
RESUMEN
Gain-of-function mutations in the transient receptor potential (TRP) cation channel subfamily C member 6 (TRPC6) gene and mutations in the NPHS2 gene encoding podocin result in nephrotic syndromes. The purpose of this study was to determine the functional significance of biochemical interactions between these proteins. We observed that gating of TRPC6 channels in podocytes is markedly mechanosensitive and can be activated by hyposmotic stretch or indentation of the plasma membrane. Stretch activation of cationic currents was blocked by small interfering RNA knockdown of TRPC6, as well as by SKF-96365 or micromolar La(3+). Stretch activation of podocyte TRPC6 persisted in the presence of inhibitors of phospholipase C (U-73122) and phospholipase A2 (ONO-RS-082). Robust stretch responses also persisted when recording electrodes contained guanosine 5'-O-(2-thiodiphosphate) at concentrations that completely suppressed responses to ANG II. Stretch responses were enhanced by cytochalasin D but were abolished by the peptide GsMTx4, suggesting that forces are transmitted to the channels through the plasma membrane. Podocin and TRPC6 interact at their respective COOH termini. Knockdown of podocin markedly increased stretch-evoked activation of TRPC6 but nearly abolished TRPC6 activation evoked by a diacylglycerol analog. These data suggest that podocin acts as a switch to determine the preferred mode of TRPC6 activation. They also suggest that podocin deficiencies will result in Ca(2+) overload in foot processes, as with gain-of-function mutations in the TRPC6 gene. Finally, they suggest that mechanical activation of TRP family channels and the preferred mode of TRP channel activation may depend on whether members of the stomatin/prohibitin family of hairpin loop proteins are present.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glomérulos Renales/metabolismo , Proteínas de la Membrana/metabolismo , Podocitos/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Bloqueadores de los Canales de Calcio/farmacología , Línea Celular , Membrana Celular/metabolismo , Clorobenzoatos/farmacología , Cinamatos/farmacología , Citocalasina D/farmacología , Diglicéridos/farmacología , Estrenos/farmacología , Guanosina Difosfato/análogos & derivados , Guanosina Difosfato/farmacología , Células HEK293 , Humanos , Imidazoles/farmacología , Péptidos y Proteínas de Señalización Intercelular , Péptidos y Proteínas de Señalización Intracelular/genética , Activación del Canal Iónico , Proteínas de la Membrana/genética , Ratones , Mutación , Síndrome Nefrótico , Péptidos/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Inhibidores de Fosfolipasa A2 , Pirrolidinonas/farmacología , Interferencia de ARN , ARN Interferente Pequeño , Ratas , Venenos de Araña/farmacología , Canales Catiónicos TRPC/genética , Canal Catiónico TRPC6 , Tionucleótidos/farmacología , Fosfolipasas de Tipo C/antagonistas & inhibidores , ortoaminobenzoatos/farmacologíaRESUMEN
Canonical transient receptor potential-6 (TRPC6) channels have been implicated in the pathophysiology of glomerular diseases. TRPC6 channels are typically activated by diacylglycerol (DAG) during PLC-dependent transduction cascades. TRPC6 channels can also be activated by reactive oxygen species (ROS). We previously showed that podocin is required for DAG analogs to produce robust activation of TRPC6 channels in podocytes. Here we show that endogenous TRPC6 channels in immortalized podocytes reciprocally coimmunoprecipitate with the catalytic subunit of the NADPH oxidase NOX2 (gp91(phox)). The NOX2-TRPC6 interaction was not detected in cells stably expressing a short hairpin RNA targeting podocin, although NOX2 and TRPC6 were present at normal levels. Application of a membrane-permeable DAG analog [1-oleoyl-2-acetyl-sn-glycerol (OAG)] increased generation of ROS in podocytes, but this effect was not detected in podocin knockdown cells. OAG also increased steady-state surface expression of the NOX2 regulatory subunit p47(phox). In whole cell recordings, TRPC6 activation by OAG was reduced in podocytes pretreated with the NOX2 inhibitor apocynin, by the pan-NOX inhibitor diphenylene iodonium, and by tempol, a ROS quencher. Cholesterol depletion and disruption of lipid rafts by methyl-ß-cyclodextrin reduced activation of podocyte TRPC6 channels by OAG and also eliminated the NOX2-TRPC6 interaction as assessed by coimmunoprecipitation. These data suggest that active NOX2 assembles with TRPC6 at podocin-organized sterol-rich raft domains and becomes catalytically active in response to DAG. The localized production of ROS contributes to TRPC6 activation by chemical stimuli such as DAG. Podocin appears to be necessary for assembly of the NOX2-TRPC6 complex in lipid rafts.
Asunto(s)
Diglicéridos/farmacología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Proteínas de la Membrana/metabolismo , NADPH Oxidasas/metabolismo , Podocitos/metabolismo , Canales Catiónicos TRPC/metabolismo , Animales , Células CHO , Línea Celular Transformada , Cricetinae , Cricetulus , Técnicas de Silenciamiento del Gen , Ratones , NADPH Oxidasa 2 , Podocitos/efectos de los fármacos , Unión Proteica/fisiología , Canal Catiónico TRPC6RESUMEN
Glomerular crescents are most common in rapidly progressive glomerulonephritis but also occur in non-inflammatory chronic glomerulopathies; thus, factors other than inflammation should trigger crescent formation, eg vascular damage and plasma leakage. Here we report that Alport nephropathy in Col4A3-deficient Sv129 mice is complicated by diffuse and global crescent formation in which proliferating parietal epithelial cells are the predominant cell type. Laminin staining and transmission and acellular scanning electron microscopy of acellular glomeruli documented disruptions and progressive disintegration of the glomerular basement membrane in Col4A3-deficient mice. FITC-dextran perfusion further revealed vascular leakage from glomerular capillaries into Bowman's space, further documented by fibrin deposits in the segmental crescents. Its pathogenic role was validated by showing that the fibrinolytic activity of recombinant urokinase partially prevented crescent formation. In addition, in vitro studies confirmed an additional mitogenic potential of serum on murine and human parietal epithelial cells. Furthermore, loss of parietal cell polarity and unpolarized secretion of extracellular matrix components were evident within fibrocellular crescents. Among 665 human Alport nephropathy biopsies, crescent formation was noted in 0.4%. We conclude that glomerular vascular injury and GBM breaks cause plasma leakage which triggers a wound healing programme involving the proliferation of parietal cells and their loss of polarity. This process can trigger cellular and fibrocellular crescent formation even in the absence of cellular inflammation and rupture of the Bowman's capsule.
Asunto(s)
Membrana Basal Glomerular/metabolismo , Membrana Basal Glomerular/patología , Nefritis Hereditaria/metabolismo , Nefritis Hereditaria/patología , Adolescente , Adulto , Animales , Autoantígenos/genética , Proteínas Sanguíneas/farmacología , Línea Celular Transformada , Polaridad Celular/fisiología , Proliferación Celular/efectos de los fármacos , Colágeno Tipo IV/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Células Epiteliales/metabolismo , Células Epiteliales/patología , Femenino , Fibrinólisis/efectos de los fármacos , Humanos , Masculino , Ratones de la Cepa 129 , Ratones Noqueados , Nefritis Hereditaria/prevención & control , Cultivo Primario de Células , Activador de Plasminógeno de Tipo Uroquinasa/farmacología , Cicatrización de Heridas/fisiologíaRESUMEN
Reactive oxygen species (ROS), which excessively arise in diabetes and systemic inflammatory diseases, modify cellular lipids and cellular lipid composition leading to altered biophysical properties of cellular membranes. The impact of lipid peroxidation on transmembrane signaling routes is not yet well studied. The canonical transient receptor potential channel 6 (TRPC6) is implicated in the pathogenesis of several forms of glomerular diseases. TRPC6 is sensitive to membrane stretch and relies on a distinct lipid environment. This study investigates the effect of oxidative alterations to plasma membrane lipids on TRPC6 activity and the function of the glomerular filter. Knockout of the anti-oxidative, lipid modifying enzyme paraoxonase 2 (PON2) leads to altered biophysical properties of glomerular epithelial cells, which are called podocytes. Cortical stiffness, quantified by atomic force microscopy, was largely increased in PON2-deficient cultured podocytes. PON2 deficiency markedly enhanced TRPC6 channel currents and channel recovery. Treatment with the amphiphilic substance capsazepine in micromolar doses reduced cortical stiffness and abrogated TRPC6 conductance. In in vivo studies, capsazepine reduced the glomerular phenotype in the model of adriamycin-induced nephropathy in PON2 knockout mice and wildtype littermates. In diabetic AKITA mice, the progression of albuminuria and diabetic kidney disease was delayed. In summary, we provide evidence that the modification of membrane characteristics affects TRPC6 signaling. These results could spur future research to investigate modification of the direct lipid environment of TRPC6 as a future therapeutic strategy in glomerular disease.
Asunto(s)
Diabetes Mellitus , Nefropatías Diabéticas , Canales de Potencial de Receptor Transitorio , Ratones , Animales , Nefropatías Diabéticas/metabolismo , Canal Catiónico TRPC6 , Canales Catiónicos TRPC/metabolismo , Doxorrubicina/efectos adversos , Ratones Noqueados , CapsaicinaRESUMEN
BACKGROUND: Targeted therapies to stabilize the clinical manifestations and prolong pregnancy in preeclampsia do not exist. Soluble fms-like tyrosine kinase 1 (sFlt-1), an alternatively spliced variant of the vascular endothelial growth factor receptor 1, induces a preeclampsia-like phenotype in experimental models and circulates at elevated levels in human preeclampsia. Removing sFlt-1 may benefit women with very preterm (<32 weeks) preeclampsia. METHODS AND RESULTS: We first show that negatively charged dextran sulfate cellulose columns adsorb sFlt-1 in vitro. In 5 women with very preterm preeclampsia and elevated circulating sFlt-1 levels, we next demonstrate that a single dextran sulfate cellulose apheresis treatment reduces circulating sFlt-1 levels in a dose-dependent fashion. Finally, we performed multiple apheresis treatments in 3 additional women with very preterm (gestational age at admission 28, 30, and 27+4 weeks) preeclampsia and elevated circulating sFlt-1 levels. Dextran sulfate apheresis lowered circulating sFlt-1, reduced proteinuria, and stabilized blood pressure without apparent adverse events to mother and fetus. Pregnancy lasted for 15 and 19 days in women treated twice and 23 days in a woman treated 4 times. In each, there was evidence of fetal growth. CONCLUSIONS: This pilot study supports the hypothesis that extracorporeal apheresis can lower circulating sFlt-1 in very preterm preeclampsia. Further studies are warranted to determine whether this intervention safely and effectively prolongs pregnancy and improves maternal and fetal outcomes in this setting.